Intermolecular single-stranded DNA (ssDNA) ligation is important for various biotechnical applications, such as LMPCR (Zhang et al., 1996, Nucleic Acids Res. 24:990-991; Dai et al., 2000, Nat. Biotech. 18:1108-1111; Yeku and Frohman, 2011, Methods Mol. Biol. 703:107-122) and cDNA library construction (Levin et al., 2010, Nat. Meth. 7:709-715; Lucks et al., 2011, Proc. Natl. Acad. Sci. USA 108:11063-11068), each of which require a fixed sequence DNA oligonucleotide to ligate to an unknown 3′-end of a cDNA. Currently, only a few protocols are available to perform such intermolecular ssDNA ligations, which use Circligase I (Lucks et al., 2011, Proc. Natl. Acad. Sci. USA 108:11063-11068; Li et al., 2006, Anal. Biochem. 349:242-246; Blondal et al., 2005, Nucleic Acids Res. 33:135-142) or T4 RNA ligase I (Zhang et al., 1996, Nucleic Acids Res. 24:990-991; Tessier et al., 1986, Anal. Biochem. 158:171-178). In addition, Circligase II recently became commercially available; however Circligase II is identical to Circligase I, differing only in the level of protein adenylation. The nucleotide preferences, referring to the likelihood of ligation to a certain base (given an equal concentration of the 4 bases in the reaction mixture), of these ligation methods on intermolecular ssDNA ligations, however, were not available prior to the present invention.
Identification and remediation of nucleotide bias in ssDNA ligation is crucial because such bias can fail to quantitatively capture the original information stored in the DNA sample. Indeed, nucleotide preference in nucleic acid ligations can potentially lead to misinterpretation of gene expression levels (Jayaprakash et al., 2011, Nucleic Acids Res. 39(21):e141; McCormick et al., 2011, Silence 2(1):2; Linsen, et al., 2009, Nat. Meth. 6:474-476). Nucleotide bias and inefficiencies of ssDNA ligation hampers ligation methods currently in use.
Thus, there is a need in the art for compositions and methods providing fast, efficient, and low-sequence bias ligation of ssDNAs. The present invention satisfies this unmet need.